Ion exchange process for separating proteins



Patented Feb. 16, 1954 PROTEIN ION EXCHANGE PROCESS FOR SEPARATIN GAllen F. Reid, Dallas, Tex.

No Drawing. Application July 6, 1951, Serial No. 235,557

6 Claims. (Cl. 260-412) This invention relates to control ofsolubilities by ion-exchange reactions and more particular- 1y tovarying salt concentrations in solutions by ion-exchange reactions tocontrol the solubility of the solution. This case is acontinuation-inpart of my co-pending application Ser. No. 20,583 filedApril 12, 1948 (now abandoned).

It is an object of this invention to adjust the ion concentration in asolution of substances by ion-exchange reactions.

It is a further object of this invention to separate substances from asolution by varying the ion concentration in the solution.

It is more particularly the object of this invention to separateproteins whose solubilities differ from one another in solutions ofvarious salt concentrations by adjusting the ion concentration of thesalts in the solutions.

The process of the present invention consists broadly in an exchange ofions between a solution and an ion-exchange resin whereby selected ionsare removed from the solution. This removal changing the concentrationof the ions in the solution affects the solubilities of the othersubstances in the solution and the difference in the solubilities isemployed to cause a separation of the substances.

Ion-exchange materials are solids which have the capacity of exchangingone ion for another in a solution contiguous to their surfaces. Forexample, if RH is the acid form of a cation-exchange resin in contactwith a solution of sodium chloride:

And if R'OH is the hydroxyl form of an anionexchange resin in contactwith a solution of sodium chloride:

of hydrogen ion in the attempt to restore equililibrium. The sodiumconcentration of the so lution is reduced at the top of the column. Thissodium depleted solution progresses further down the bed, its sodiumconcentration appreaching nearer and nearer to 0.1 M until it exits atthat concentration. More and more solution can be put through and haveits concentration adjusted to 0.1 M until such time as the bed capacityfor sodium under the operating conditions has become saturated farenough down so that the remaining layers of resin are not able to removeall of the excess sodium. A similar situation exists if theconcentration of sodium in the solution introduced had been less than0.1 M-sodium from the resin going into the solution to make up thedifference.

Similarly, an anion-exchange resin bed could adjust the concentration ofany anions in a solution progressing through it. Thus with a mixture ofthe two types of ion-exchangers, a solution can be adjusted toconcentrations of ions compatible with the capacity of the exchangers,if, prior to passage of that solution, the bed has been conditioned tobe in equilibrium with the ion concentration desired.

Many natural proteins are soluble in aqueous solutions of commonchemical compounds and are ofttimes found in mixtures in the samesolutions from which one or several of them is desired to be removed. Asan example, we may consider the fractions of human blood plasma. Theseinclude proteins known as albumins which are soluble in distilled water,and various types of globulins which are not soluble in distilled waterbut are soluble in various concentrations of solutions of sodiumchloride. When human blood serum is separated from the blood cells andfibrin, it contains these proteins in solutions which have a saltconcentration of approximately 0.17 mole per liter. The globulinspresent can be precipitated and the albumin left in the solutions ifmost of the salts are removed from this serum. Such fractionation isvaluable because the separated albumin fractions can be used medicinallyfor the treatment of shock, etc. and the separated globulin containsseveral fractions which are medicinally useful as immune sera and in thetreatment of hemophilic cases, etc. Be-. cause of the differentsolubilities of these components, it is evident that fractionation canbe attained by adjusting the concentration of the non-proteinconstituents of the solutions. Up to the present time no practicalmethod has been advanced for adjusting those concentrations for thatpurpose which was satisfactory from the standpoints of expense and theprotection of the chemically and thermally sensitive products. In orderto adjust the concentration of these chemical constituents, i haveintroduced the solution at conservative temperatures to beds ofion-exchange resins which have been conditioned so that they will absorbions in the proper amounts and proportions to leave a concentration ofions in the solutions favorable for such fractionation.

The introduction of such an improved method for this fractionation isimportant since present methods, as well as being quite expensive,involve processes such as the use of very cold alcohol which decreasesthe potency and desirability of some of the products.

The following is a procedure which may be followed in one application ofthe principle of this invention:

A mixture of a cation-exchange resin and an anion-exchange resin iswashed thorougly with distilled water to reduce the salt concentration.This mixture of resins is placed in a series of columns and washedagain. The solution of proteins to be separated, such as defibrinatedblood plasma, is poured through these prepared columns. At the end ofthis treatment the ions which are taken bythe resins in the ion-exchangereactions are removed from the. solution. The removal of these ions bychanging the ion concentration of the solution changes the solubility ofone or more of the proteins in the solution with relation to the otherproteins and consequently causes precipitation. The different proteinsare precipitated at different points in the process as theirsolubilities are different in the various ion concentrations.

Applying this to human blood plasma, by removing salt from the plasma,the globulins are rendered insoluble in the solution and precipitated,leaving the albumin which is soluble in distilled water. Theprecipitated globulins may be centrifuged off from the treated solutionand those which remain trapped in the columns dissolve out with a salinesolution. The final solution which has passed through all the bedscontains no appreciable globulins after centrifugation. Albumin which isleft in the precipitated globulin fractions may be dissolved out withdistilled water without dissolving the globulins. The globulin fractionswhich have been Washed out of the columns with the saline solution maycontain some albumin because of the hold-up of liquid in the columnsunder normal operation.

The globulin fractions are separated from each other by a process ofpreferential leaching and precipitation. The mixture is first washedfree of albumin and then leached with solutions of sodium chloride at acontrolled pH. Under these conditions one globulin sub-fraction isdissolved preferentially from the others. The salt concentration and thepH of the leaching solution is then adjusted with an ion-exchange resinmixture and a selective precipitation of globulins is obtained. Thisglobulin precipitate is subjected to another selective leaching followedby a selective precipitation. In this way, using proper control of thepH, the sodium chloride concentration and the concentration of othersalts, it is possible to fractionate the globulins to any degree ofrefinement. In the described separation of globulin and albumin in bloodplasma the mixture of two types of resins used is intended to keep thepH of the blood plasma solution neutral between alkalinity and acidity.This is achieved by having the cation-exchange resin remove the sodiumion in this separation replacing it with hydrogen ion and theanionexchange resin removing the chloride ion replacing it with ahydroxyl ion. The subsequent neutralization of the hydroxyl ions withthe excess hydrogen ions and vice versa insures that no highconcentration of acid is present in the solution. This is important asan acid pH would cause precipitation of the albumin and possiblydenaturation of the proteins, and a basic pH would denature theproteins. The other blood serum salts are removed similarly. In thepassage of the solutions through the ion-exchange resins some of theanion-exchange resin may be dissolved and taken into the solution.Pyrogens which are contaminants formed by bacteria may be present in thereconstituted plasma and could be present in the distilled water whichis used in the operations. Any bacterial matter which might also bepresent could. form other pyrogens unless the operations were carriedout at an elevated temperature which would be objectionable as causingdenaturation of the proteins. Therefore, to remove these impurities andcontaminants. from the solution it is passed through two final bedscontaining only the cation-exchange resin. These beds are conditionedfirst with distilled water and finally with pyrogen-free water and thusare able to take up the aforementioned contaminants. Finally, thesolution is sent through a bacterial filter to effectively remove anysmall particles of resin or bacteria which might have been picked up.This is removed by reprocessing. The albumin in the solution afterconcentration safe for intravenous use.

The following example will serve to illustrate this process:

I. A mixture of four parts of Amberlite IR-lOO H, a cation-exchangeresin, furnished by the Resinous Products Chemical Company ofPhiladelphia, and 9 parts of Permutit Deacidite, an anion-exchangeresin, furnished by the Permutit Company of New York city, Was washedthoroughly with distilled water until the salt concentration was lessthan four parts per million. This mixture of resins was put in a seriesof 5 columns, each about 2 inches in diameter and 4 feet in length, andwashed again. 1500. cc. of double strength reconstituted defibrinatedblood plasma were poured consecutively through these columns. At the endof this treatment, the total salt to albumin ratio was less than 1 to1000 in the albumin solution. Amberlite iii-10o H is aphenol-formaldehyde resin with a polyhydric phenol base and cationicallyactive --SO3H groups. Deacidite is a highly basic aliphatic amine typeanion exchange resin. Arnberlite IR- i B is an amine type resinous anionexchanger containing approximately 14% nitrogen in the hydroxyl form.

A further application of the process of this invention is shown inremoving hemoglobin from samples of blood serum. In samples of bloodserum a certain amount of hemoglobin which is released by the fractureof red blood cells is present and soluble in salt-free solutions. It isdesirable to remove the hemoglobin, a highly colored impurity, from thealbumin fractions. Hemoglobin dissociates in a solution of sodiumsalicylate at values of pH which will not precipitate albumin. Byadjusting the concentration of sodium salicylate and acid ion to causedissociation of the hemoglobin and then removing the iron-containing ionsalts, the sodium salicylate and acid with a mixture of cation andanionexchange resins, the hemoglobin is prevented from reassociating andits objectionable nature is eliminated.

The following example serves to illustrate this process:

II. A solution of blood serum albumin was made 0.1 molar to sodiumsalicylate and then acidified to a pH of about 5.5. This dissociated thehemoglobin in the blood serum solution. The treated solution was thenpassed in contact with a mixture of 9 parts Deacidite and 4 partsAmberlite IR 100 H. The hemoglobin was reduced in the solution by afactor of more than 100 to less than 3 parts hemoglobin per 10,000 partsof albumin.

Another application of this invention is the separation of some of theproteins in milk. Here a sample of concentrated milk whey containinglactoalbumin and lactoglobulin may be passed through a washed mixture of70% Zeocarb H, an organic cation-exchange resin of the coal derivativetype, furnished by the Permutit Company and 30% Amberlite IR 43, anamine type resinous anion exchanger containing approximately 14%nitrogen in the hydroxyl form, furnished by the Resinous ProductsChemical Company. The salt-free solution contains primarilylactoalbumin, globulins being precipitated out as the salt concentrationgoes down.

Another example is in the separation of the globulins from the albuminin bovine serum. This is important as bovine albumin is routinely usedin the medical sciences as a diagnostic aid. This separation may beaccomplished by using either a mixture of four parts of Amberlite IR100, a phenol-formaldehyde resin with a polyhydric phenol base andcationically active- SOsH groups, and nine parts of Permutit Deacidite,a highly basic aliphatic amine type anionexchange resin; or by using amixture of one part of Amberlite IR 100 and two parts of Amberlite IRA400, a strongly basic anion-exchange resin with a high stability. Whenthe concentration of the salts has been brought down to an ionicstrength of .015 by treating the solution with a resin mixture, most ofthe globulins precipitate out leaving a solution which may besatisfactorily purified by a subsequent heat treatment.

Another application of the invention is the separation of proteinsproduced by the action of bacteria on culture media. These proteinproducts may be used, when modified somewhat chemically, as antigens toproduce immunity in the body because of their stimulation of theproduction of antibodies. An example of this is diphtheria toxoid. Inthe production of this toxoid, Corynebacterium dipht eriae is allowed togrow in a culture medium. The toxicity of the resulting protein isdestroyed by treatment with formaldehyde or some other modifying meanswhile still preserving the antigenicity of the material. This detoxifiedprotein must then be freed from other colloids present in order to makea preparation which is satisfactorily active and safe for injection. Itis found that after the gross impurities have been separated from thetoxoid, it may be further purified by subjecting its solution to amixture of two parts Amberlite IRA 400 to one part Amberlite IR 100,thus reducing the salt concentration to below an ionic strength of .001while keeping the pH value between 6.5 and 7.5. This treatment causesprecipitation of contaminating colloid material which may then bemechanically separated from the rest of the proteins. The resultingprotein may be even further separated by adjusting'the pH with AmberliteIR to a value of 313 where an electrophoretically pure component isprecipitated out. This may then be separated from the rest of thesolution by mechanical separation. A type of antigen may be produced bysubjecting certain bacteria to other influences. One such method is tosubject bacteria to ultrasonic vibrations. However, more than one typeof antigen may be produced by such treatment. For certain medicalreasons it is often advisable to separate these. Another application ofthe invention is the separation of such substances. As an example ofthis, a colloidal suspension of the antigens from the sonic disruptionof Staphylococcus aureus with an ionic strength of 0.10 and a pH of 7was treated with one-third its volume of a mixture of one part of thecationexchange resin Amberlite IR and two parts of the anion exchangeresin Amberlite IRA 400 for five minutes. This reduced the solution toan ionic strength of 0.001 and caused the precipitation of one proteinfraction leaving another protein fraction in solution. In the treatmentthe pH was kept in the range from 6-7 avoiding alkali or aciddenaturation of the proteins. The precipitated fraction was redissolvedin a solution of ionic strength 0.20 and the two portions contained thebiologically difierent types of antigen.

As another modification of the procedure of the application of thisinvention, the reduction of the salt concentration of the solution ofdiphtheria toxoid may be accomplished by first subjecting the solutionto a small amount of Amberlite IRA 400, not allowing the pH to riseabove 8, then transferring the solution to a small amount of AmberliteIR 100, not allowing the pH to go below 6, then transferring thesolution to another batch of IRA 400, then to another batch of IR 100and so on, always keeping the pH between the limits of 6 and 8 until theionic strength has been reduced to- .001. This treatment causesprecipitation of contaminating colloid material as in the example above.

A similar application of the invention exists in the purification oftetanus toxoid. After production in a similar manner by the action ofClostrzdz'um tetani on a nutrient medium and gross separation of theprotein, the toxoid containing fraction may be purified from other protein colloids by removal of the salts to an ionic strength of 0.001,keeping the pH value between 6.5 and 7.5 using a mixture of two parts ofAmberlite IRA 400 to one part Amberlite IR 100. The precipitatedimpurities may be removed by mechanical separation. The remainingsolution may be further separated by reducing the pH with Amberlite IR100 to 5.0, at'which time an electrophoretically discrete proteincomponent will precipitate out. This may be removed by mechanicalseparation.

As another modification of the procedure of the application of thisinvention, the reduction of the salt concentration of the solution oftetanus toxoid may be accomplished by first subjecting the solution to asmall amount of Amberlite IR 100, not allowing the pH to go below 6,then transferring the solution to a small amount of Amberlite IRA 400,not allowing the pH to rise above 8, then transferring the solution toanother batch of IR 100, then to another batch of IRA 400 and so on,always keeping the pH between the limits of 6 and 8 until-the ionicstrength has been reduced to .001. This treatmay be accomplished by mentcauses precipitation of contaminatin colloid material as in the exampleabove.

Two important by-products of the meat packing industry are the enzymespepsin and rennin. They occur in the gastric secretions of such animalsas cattle and hogs and are recovered from these sources. In the state inwhich they are recovered in the crude secretions they are not practicalfor commercial sale or usage. It is therefore necessary to purify them,A further application of this invention may be utilized in thispurification. It is found that by using a mixture of about one partAmberlite IRA, 400 to one part Amberlite IR 100 for the removal of saltsand acid to an ionic strength of .001 and a pH of 3, many of the proteinimpurities are precipitated out from a pepsin solution. These may beremoved by mechanical separation. On further treatment of the pepsinsolution with Amberlite IRA 400 to bring the pH to 3.52, veryconcentrated pepsin will precipitate out. The mixture of .Amberlites IRA400 and IR 100 serves to keep the pH low enough .so that the pepsin,which is unstable at a high pH, is not denatured.

As another modification of the procedure of the application of thisinvention, the reduction of the salt concentration of the solution ofpepsin may be accomplished by first subjecting the solution to a smallamount of Amberlite IR 100, not allowing the pH to go below 1, thentransferring the solution to a small amount of Amberlite IRA 400, notallowing the pH to rise above 3, then transferring the solution toanother batch of IR 100, then to another batch of IRA 400 and so on,always keeping the pl-l between the limits of 1 and 3 until the ionicstrength has been reduced to 0.001. The solution is finally treated withAmberlite IRA 400 to bring the pH to 3.52. This treatment causesprecipitation of concentrated pepsin as in the example above.

In a similar application of the invention, if a crude rennin solution ata neutral pH is treated with a mixture of two parts Amberlite IRA 400and one part Amberlite IR 100, the salt concentration may be brought toan ionic strength of i .001 keeping the pH between 6.0 and 7.5. Manycontaminating proteins are precipitated upon such treatment and may beremoved by mechanical separation. Upon reduction of the pH to 3.75 bytreatment with Amberlite IR 100, a concentrated rennin is precipitated.

As another modification of the procedure of the application of thisinvention, the reduction of the salt concentration of the solution ofrennin first subjecting the solution to a small amount of Amberlite IR100, not allowing the pH to go below 5, then transferring the solutionto a small amount of Amberlite IRA 400, not allowing the pH to riseabove 8, then transferring the solution to another batch of IR 100, thento another batch of IRA 400 and so on, always keeping the pH between thelimits of 5 to 8 until the ionic strength has been reduced to .001.Finally the solution is brought to a pH of 3.75 by treatment withAmberlite IR 100. This treatment causes precipitation of contaminatingcolloid material as in the example above.

In the above described preparations, the enzymes retained their originalactivity. In other methods of removal or separation, such as theprecipitation by salting-out with a high concentration of sodiumsulphate, these enzymes may be denatured.

Many natural proteins are of vegetable origin and are quite important inthe economic life of today, particularly in the food industry. Forexample, it is found that the type of proteins found in ordinary wheatflour are very important in the use of this flour for the manufacture ofproducts such as bread, cake, etc. Attempts to modify the concentrationsof the proteins in flour so as to be able to market it for certain useshave been defeated by the failure to separate out some fractions fromother protein constituents by a prac tical means. An application of thisinvention to this separation is to solubilize a portion of the wheatproteins in a salt solution and then to precipitate out certain proteinsby removal of the salts using ion exchange resins. In a specificinstance some common wheat flour was mixed with a solution of salt withan ionic strength of approximately 0.15. The resulting solution of wheatproteins was mechanically separated from the remaining solid materialand subjected to a mixture of twov parts Amberlite IRA 400 and one partof Amberlite IR 100. As the salt concentration decreased with the pHremaining between 6.5 and 7.5, wheat proteins fractionally precipitatedout. These could be mechanically separated from the solution.

Other natural vegetable proteins of considerable food value are found inpeanuts. After the oil was removed from some crushed peanuts, some ofthe proteins were dissolved in a salt solution of an ionic strength ofapproximately 0.60. lhese were then fractionally precipitated byreducing the salt concentration to an ionic strength of less than .01using a mixture of two parts of Amberlite IRA 400 to one part ofAmberlite IR thus keeping the pH at a value of 6.5 to 7.

Certain vegetable proteins have enzymatic activity. One of the mostimportant of these is urease which is made from iackbeans. After thisprotein has been separated grossly from the inactive material accordingto the most advanced means in common practice, the enzyme is stillcontaminated with much protein which serves not only to dilute itsactivity but also actually to inhibit some of the enzyme action. If someof this grossly purified urease is placed in a solution of salt with anionic strength of .02 and then subjected to a mixture of two parts ofAmberlite IRA 400 to one part of Amberlite IR 100 and the saltconcentration is brought to an ionic strength of .001 with the pHremaining between 6.5 and 7.5, the urease will be precipitated in a muchpurer form and its total activity when used will be increased by severalhundred percent.

Another important vegetable enzyme is papain. Here again, theconventionally purified papain preparation is a mixture of severalproteins. These can be separated from each other by putting them intosolution in a salt solution of an ionic strength of 0.02. As the saltconcentration is decreased to an ionic strength of 0.001 while keepingthe pH between 7.5 and 8 by treating the solution with a mixture of onepart of Amberlite IR 100 to 3 parts of Amberlite IRA 400, contaminatingimpurities precipitate out leaving a purer preparation of papain.

Still another application of the method of this invention is a resinartificial kidney. The kidney normally serves to remove waste productssuch as urea, etc. from the blood stream. If its functioning is impairedtoo much, even temporarily, the animal dies. However, by conditioning abed containing a mixture of 9 parts Amberlite IR 100 H and one partDeacidite with a solution of approximately the same ionic concentrationsas normal blood serum, and then passing blood from dogs with their.kidneys removed through the bed and back into the dogs it was possibleto reproduce the purifying efiect of the normal kidneys. A refinementincluded addition of a small bed of Amberlite IR 100 H at the exit endof the large bed to absorb the small amounts of dissolved Deacidite. Thepurified blood showed no detectable deleterious effects.

It can also be seen that the same process can be used in individualoperations for direct application. Globin was prepared from hemoglobinby treating an aqueous hemoglobin solution with sodium salicylate andhydrochloric acid and then a resin mixture as above described. Pyrogenswere removed from old distilled water by passing the water through aresin bed that had previously been washed with pyrogen-free water.

Although only Amberlite IR 100 H, Amberlite IR 413, Zeocarb H andDeacidite have been used to illustrate the application of the process ofthe present invention to the removal of ions from a protein solution,other ion-exchange resins may be used successfully in the presentprocess.

The collective singular of a heterogeneous substance such as globulin,protein or albumin conventionally also covers the plural in accordancewith established usage.

Protein separation processes adjusting the pH to values outside of theneutral zone disclosed and not claimed herein are disclosed and claimedin applicant's later filed application Ser. No. 392,507.

I claim:

1. In a process of separating a protein from other proteins in anaqueous solution containing a plurality of proteins as solutes and alsohaving a salt content present as cations and anions in a concentrationrendering said first-mentioned protein soluble in said solution and inthe absence of which cations and anions said protein is insoluble insaid solution, another protein solute of said solution being solubletherein in the absence of said cations and anions in said concentration,the steps of providing a mixture of cation-exchange and anion-exchangeresins conditioned to be in equilibrium with an ion concentration atwhich said solution-contained cations and anions are removed from saidsolution, passing said aqueous solution of said proteins and salt solutein contact with said cationexchange resin and said anion-exchange resinadjusting the pH of the solution to substantially neutral in the range 6to 8 between alkalinity and acidity by ion-exchange between saidsolution and said resins, removing from said solution said solubilizingcations by ion exchange with said cation-exchange resin to reduce saidsolubilizing cations and removing from said solution said solubilizinganions by ion exchange with said anion-exchange resin to reduce saidsolubilizing anions, and thereby precipitating said first-mentionedprotein from said solution of said proteins, and separating saidprecipitate from said solution.

2. A process of separating a protein from other proteins in an aqueoussolution as set forth in claim 1 in which the aqueous solution isselected from the group consisting of human blood plasma, human bloodserum, milk, bovine blood serum, 2. solution of diphtheria toxoid, acolloidal suspension of antigens, a solution of tetanus toxoid, a cruderennin solution, a solution of wheat proteins, a solution of peanutproteins, a solution of urease, and a solution of papain.

3. In a. process forthe separation of certain proteins from otherproteins in a blood solution selected from the group consisting of bloodplasma and blood serum and aqueous solutions of natural blood proteinsand containing an adequate concentration of the natural anion and cationsolubilizing ions, the said certain proteins being solubilized by saidions in the blood solution and the other said proteins being bothsoluble in the blood solution and in the absence of said concentrationof said solubilizing ions, the steps of providing an anion exchangeresin and a cation exchange resin which are capable of removing saidsolubilizing ions from said blood solution, passing said solution incontact with said anion exchange resin and said cation exchange resin,adjusting the pH of said blood solution to neutral by ion exchange withsaid anion exchange resin and said cation exchange resin, removing froma portion of said solution both said anion solubilizing ions and saidcation solubilizing ions from said Iblood solution by ion exchange withsaid anion exchange resin and said cation exchange resin to provide areduction of both said anion and cation solubilizing ions in saidportion thereby precipitating therefrom said certain proteins in theblood solution and then separating the precipitated porteins from saidsolution retaining the said other proteins.

4. In a process for the separation of globulins from other proteins inan ion-containing natural blood solution selected from the groupconsisting of blood plasma and blood serum and aqueous solutions ofnatural blood proteins in which said globulins and other proteins aresoluble, said globulins being insoluble in the absence of an adequateconcentration of said ions, the steps of providing an anion exchangeresin and a cation exchange resin which are capable of removing saidsolubilizing ions from said blood solution, passing said blood solutionwith both said globulins and other proteins in contact with said anionexchange resin and said cation exchange resin, adjusting the pH of saidblood solution to neutral by ion exchange between said solution and saidresins, removing from said solution said anion solubilizing ions by ionexchange with said anion exchange resin to provide a reduction of saidanion solubilizing ions in said solution, removing cation solubilizingions from said solulected from the group consisting of blood plasma andblood ALLEN F. REID. (References on following page) 2 References Citedin the file of this patent Steinberg, Proc. Soc. Exptl. B101. and MadUNITED TATES PATENTS (June 1944:), pp. 124*127.

S Myers et ah, Ind. and Eng. Chem. (May 19411 Number Name Date VOL 43,pp.

2340316 HOlmFS 1941 5 (John et 21., J. Am. Chem. Soc., Mar. 1946, vol2,275,219 Urbaln et a1 Mar. 3, 1942 3 459 475 2,461,505 Daniel Feb. 15,1949 OTHER REFERENCES

1. IN A PROCESS OF SEPARATING A PROTEIN FROM OTHER PROTEINS IN ANAQUEOUS SOLUTION CONTAINING A PLURALITY OF PROTEINS AS SOLUTES AND ALSOHAVING A SALT CONTENT PRESENT AS CATIONS AND ANIONS IN A CONCENTRATIONRENDERING SAID FIRST-MENTIONED PROTEIN SOLUBLE IN SAID SOLUTION THEREINAND IN THE ABSENCE OF WHICH CATIONS AND ANIONS SAID PROTEIN IS INSOLUBLEIN SAID SOLUTION, ANOTHER PROTEIN SOLUTE OF SAID SOLUTION BEING SOLUBLETHEREIN IN THE ABSENCE OF SAID CATIONS AND ANIONS IN SAID CONCENTRATION,THE STEPS OF PROVIDING A MIXTURE OF CATION-EXCHANGE AND ANION-EXCHANGERESINS CONDITIONED TO BE IN EQUILIBRIUM WITH AN ION CONCENTRATION ATWHICH SAID SOLUTION-CONTAINED CATIONS AND ANIONS ARE REMOVED FROM SAIDSOLUTION, PASSING SAID AQUEOUS SOLUTION OF SAID PROTEINS AND SALT SOLUTEIN CONTACT WITH SAID CATIONEXCHANGE RESIN AND SAID ANION-EXCHANGE RESINADJUSTING THE PH OF THE SOLUTION TO SUBSTANTIALLY NEUTRAL IN THE RANGE 6TO 8 BETWEEN ALKALINITY AND ACIDITY BY ION-EXCHANGE BETWEEN SAIDSOLUTION AND SAID RESINS, REMOVING FROM SAID SOLUTION SAID SOLUBILIZINGCATIONS BY ION EXCHANGE WITH SAID CATION-EXCHANGE RESIN TO REDUCE SAIDSOLUBILIZING CATIONS AND REMOVING FROM SAID SOLUTION SAID SOLUBILIZINGANIONS BY ION EXCHANGE WITH SAID ANION-EXCHANGE RESIN TO REDUCE SAIDSOLUBILIZING ANIONS, AND THEREBY PRECIPITATING SAID FIRST-MENTIONEDPROTEIN FROM SAID SOLUTION OF PROTEINS, AND SEPARATING SAID PRECIPITATEFROM SAID SOLUTION.